Field of the Invention
This invention relates to methods and compositions for selecting biological molecules such as nucleic acids and proteins using coated ferromagnetic density particles (FMP) or density particles (DP) and kits for research, therapeutic and diagnostic uses. More specifically, the invention relates to selection of biological molecules which are not cells, but which include components of cells such as nucleic acids, organelles, proteins, lipoproteins, glycoproteins, peptides, components present in serum or plasma, whether or not of plant, animal, or other living organisms, and similar like substances. Further, the invention relates to large scale chromatographic isolation of proteins including applications in Affinity Chromatography and High Performance Liquid Chromatography.
Description of the Related Art
In the biochemical manipulation of cells and biological molecules (such as but not limited to nucleic acids and/or proteins) it is often desirable to isolate specific cells, specific nucleic acid sequences or specific proteins from complex mixtures for downstream processing using solid phase supports as described herein. Numerous magnetic particle based cell separation technologies based on superparamagnetic particles and ferromagnetic particles exist. Separation of cells by gravity settling using dense particles has also been described. However, in the isolation of biological molecules (nucleic acids and/or proteins) only technologies based on superparamagnetic particles have been described with some success, particularly in DNA applications. To date the use of ferromagnetic particles and dense particles has been confined to the field of cell separation. Some prior literature has suggested that ferromagnetic materials may be used to separate biological molecules, but no specific details have been provided as to how this might be done. More specifically, the mentioned ferromagnetic materials are generally non-specific in nature and could not be used to isolate specific biological molecules.
On the other hand, use of superparamagnetic particles to isolate proteins is well documented. Particles commercially available from Dynal Biotech, as referenced in its 2003 Product catalog have been used for immunoprecipitation and co-immunoprecipitation of pure proteins; to pull down large protein complexes that tend to be broken down by traditional column chromatography techniques; for depletion of proteins; use of GST-fusion proteins applying anti-tag antibodies; immobilization of active enzymes used in production of chemical compounds and foods; to immunoselect proteins for further separation on SDS PAGE and downstream analysis by mass spectrometry; in MALDI-TOF mass analysis; and in various diagnostic applications including immunoassays and bioassays.
Use of superparamagnetic particles to isolate nucleic acids (RNA and DNA) is also well documented. Particles available form Dynal Biotech have been used for the isolation of pure intact mRNA from pure cell populations; mRNA for cDNA libraries; mRNA for RT-PCR and real-time PCR; mRNA for northern blotting; mRNA for microarrays. Such particles have also been used for the isolation of DNA from bacteria and blood and other clinical specimens; for tissue typing; for mutation detection and SNP's analysis. These particles have also been used for isolating RNA and DNA binding proteins using sequences bound to particles that recognize the binding region in the binding protein.
One commonly used solid support for isolating biological materials are superparamagnetic particles that come in various sizes and can be either non-uniform such as those commercially available from Advanced Magnetics, and described in U.S. Pat. No. 4,672,040. Another type of particle are those which are very uniform such as those available from Dynal Biotech and disclosed in U.S. Pat. No. 5,512,439. Other manufacturers of superparamagnetic particles include Miltenyi Biotech, Immunicon Corporation, R and D Systems, Polysciences and Stem Cell Technologies, Inc. The magnetic particles are generally <5 micron in diameter and have a density<1.8 g/cm3.
Another magnetic particle has been described in the literature. These particles are known as ferromagnetic (FMP) particles, are available commercially from TRC Biotech and are described in U.S. Published Application No. 2004-0023222. FMP have been shown to offer advantages over superparamagnetic particles in the field of cell separation. The advantages of FMP for cell separartion applications center around the core particle material, nickel, and three properties of nickel: Density: Nickel has a density of 8.9 g/cm3 which is ˜8 times the density of cells and ˜6 times the density of current magnetic particles. This difference in density results in very high mixing efficiency which in turn yields very rapid reaction kinetics often on the order of seconds; Magnetics: Because FMP are composed of solid Nickel, the particles are ferromagnetic. The ferromagnetic nature of the particles results in separation times up to ten times faster than current technologies that are based on superparamagnetic particles; and Particle Surface: Non-targeted cells do not stick to FMP because of the characteristics of the metallic particle surface. The particle is uncoated and thus is not composed of organic coatings as is seen with many superparamagnetic particles. As a result only targeted cells are captured.
While U.S. Published Application 2004-0023222 speculates generally about the use of the particles described therein in separating biological molecules, the materials described therein are non-specific to biological molecules and cannot be used to bind specific biological molecules if used in the form described therein. There is no specific disclosure in that application of how such separation of biological molecules might be done with the particles described therein. The comments are merely speculative and later tests have shown that the particles described in the published application cannot be used to separate biological molecules.
In order to more fully appreciate the differences between superparamagnetic particles and ferromagnetic particles, it is important to compare their properties as discussed hereafter.
More specifically, U.S. Pat. Nos. 5,411,863 and 5,466,574 teach that superparamagnetic particles are particles of choice for biological selection applications. Superparamagnetic materials have in recent years become the backbone of magnetic selection technology in a variety of health care and bio-processing applications. Superparamagnetic materials are highly magnetically susceptible, i.e., they become strongly magnetic when placed in a magnetic field but rapidly lose their magnetism when the magnetic field is removed. This property makes it easy to isolate and resuspend biological molecules when the magnetic field is removed.
Superparamagnetism occurs in ferromagnetic materials when the crystal diameter is decreased to less than a critical value. Such materials, regardless of their diameter (about 25 nm to about 100 microns) have the property that they are only magnetic when placed in a magnetic field. The basis for superparamagnetic behavior in ferromagnetic materials is that such materials contain magnetic material in size units below about 20 to about 25 nm, which is estimated to be below the size of a magnetic domain. A magnetic domain is the smallest volume for a permanent magnetic dipole to exist. Ferromagnetic materials, as contrasted to superparamagnetic materials, are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed. Ferromagnetism occurs only when unpaired electrons in the material are contained in a crystalline lattice thus permitting coupling of the unpaired electrons. The prior art teaches that ferromagnetic particles with permanent magnetization have considerable disadvantages, for example, as discussed in U.S. Pat. No. 5,411,863. and U.S. Pat. No. 5,466,574 for applications in biological molecule selection, since suspensions of these particles easily aggregate following exposure to a magnetic field due to their high magnetic attraction for each other. For this reason ferromagnetic particles have not been used for biological (nucleic acid selection/protein selection) applications.
In a further development as discussed in Published Application No. 2004-0023222, FMP have been shown to retain the positive attributes of superparamagnetic particles seen when applied to cell separation, and FMP eliminate the properties of superparamagnetic particles that impede their utility.
Studies using a Coulter N4 particle analyzer have shown, remarkably, that FMP of diameters from 0.05 to 1.5 micron can be dispersed by vortexing after exposure to a magnetic field (Table). This property was formerly attributed only to superparamagnetic particles.
ConditionParticle Diameter (micron)Pre-magnet1.72Post Magnet-vortex1.62Post Magnet-inversion4.96
The high magnetic susceptibility of ferromagnetic particles, as compared to superparamagnetic particles, provides rapid magnetic collection on the order of seconds to minutes. Also, because of the magnetic properties of FMP, the procedure does not require elaborate equipment.
Finally, in the field of cell separation a desirable attribute of uncoated, metallic FMP is the almost complete lack of non-specific binding of non-targeted cells. Quantitative recovery of non-targeted cells is possible using FMP. Particles labeled with CD15-antibody were used to deplete greater than 99% of the granulocytes, and a subset of monocytes known to be CD15 positive, from whole blood with quantitative recovery of the non-targeted lymphocytes (see table).
Lack of Binding to Non-Targeted Cells
LymphocytesMonocytesGranulocytesWhole Blood12,694+266523,230CD15 depleted12,815 (0)*2196 (17.5)206 (99.1)*Percent Depleted
Solid phase microparticles that separate targeted from non-targeted populations on the basis of gravity rather than magnetics have also been described, for example, in U.S. Pat. No. 5,576,185. Currently, particles that are separated based on gravity are relatively dense and large with diameters between the range of approximately 3 to 10 microns. A known feature of these particles is that because of the density difference between particles and cells, end-over-end mixing allows the particles to pass through a substantial portion of the fluid sample in which the particles are used. The particles traverse past the cells of interest and in doing so bind to the targeted cell population without non-specifically binding to non-target cells. This leads to an efficient separation and a high recovery of non-targeted cells. The separation and recovery of non-targeted cells is superior to that found with superparamagnetic selection alone.
These dense particles are designed to settle by gravity both as a mixing manner (discussed above) and as a manner to separate the desired population of cells from the remainder of the cell suspension. In fact, previous descriptions for example as discussed in U.S. Pat. No. 5,576,185, teach away from the use of smaller particles in gravity selection. For example, the disclosure of this patent teaches that superparamagnetic particles are intended to be maintained in suspension in the sample and consequently are designed for very slow or substantial elimination of gravity settling in the sample suspension. Typically, well-coated materials below 150 nm will show no evidence of settling for as long as 6 months and even longer, for example, as discussed in U.S. Pat. No. 5,622,831. Thus, superparamagnetic particles are not applicable for use in gravity selection technology or density difference mixing. Both procedures function optimally at a density difference of at least 2-3 fold between the particles and the target biomaterial when capturing cells and settling by gravity.
Gravity separation addresses several drawbacks inherent in magnetic separation procedures that utilize superparamagnetic particles including non-specific cell loss due to trapping, time of magnetic collection when using colloidal particles, and/or the high magnetic gradients required for collection of colloidal particles.
One area where superparamagnetic particles have not been used is in large scale chromatographic procedures for the purification of proteins and nucleic acids because such magnetic particles are composed of superparamagnetic materials which are not sufficiently magnetic and thus cannot function in large scale (large volume) applications. An example of large scale chromatographic isolations includes but is not limited to Affinity Chromatography.
By the term “Affinity Chromatography” is meant the purification of a biological molecule with respect to the specific binding of that biological molecule due to its chemical structure to a solid phase. With this structure, the biological molecule can reversibly bind to a reactant which has formed a covalent or non-covalent bond with a chromatographic bed material.
Many different types of chromatography are available for the purification of target proteins (recombinant or native) from different sources. For most proteins, multiple purification methods are needed to purify a target protein to homogeneity. For large-scale bioprocess purifications, certain chromatographic methods are used. These chromatography methods include:
Ion Exchange—separation of proteins by charge differential where chromatography resins are bound with molecules that are positively charged (to bind negatively charged proteins) or negatively charged (to bind positively charged proteins). Every protein has an Isoelectric Point (pI), which can be exploited to separate proteins by making them positively or negatively charged species depending on the pI of the protein. For example, a protein with a pI of 4 will be negatively charged in a buffer of a pH of 7. The protein mixture can be passed through an anion exchange column, and negatively charged proteins are bound to the column (including the target protein). The bound proteins can then be eluted off of the column by either lowering the pH of the buffer passing through the column (removing the charge from the bound proteins) or by eluting with salt containing buffer, which displaces the bound proteins. Ion exchange is normally used as a 1st step in protein purification. (1)
Affinity Chromatography—many different types of affinity chromatography are available. Types of affinity chemistries available range from general affinity (e.g. metal affinity) to more specific affinity, such as antibody/antigen, enzyme/substrate (analog), antibody/Protein A/G and receptor/ligand affinity. These methods rely on binding between the affinity matrix and the target protein. Most of the affinity interactions are highly specific for a particular target protein, and as a result, the target protein binding is both specific and tightly bound. Elution of the target protein requires harsh conditions (including low/high pH) to break the affinity interactions. Metal affinity, which utilizes a protein's affinity for specific bivalent metal ions (Cu, Ni, Co, Zn) requires more gentle conditions for elution of the target protein, including pH 6.0 elution, and competitive elution with imidazole. (2)
Affinity chromatography is a preferred choice for large-scale purification protocols, since there is little to no non-specific binding to contaminating proteins. In addition, many recombinant proteins are expressed with either affinity tags (i.e. short specific peptide sequences for use in either metal or antibody affinity purification) or as a fusion protein with the fusion protein used in the affinity purification. After purification, the fusion protein is cleaved from the target protein.
Another purification procedure is High Performance Liquid Chromatography. By the term “High Performance Liquid Chromatography” (HPLC) is meant a form of column chromatography used frequently for the isolation of biological molecules. The sample is forced through a column by liquid at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to spread out within the column, leading to broader peaks. Less time on the column then translates to narrower peaks in the resulting chromatogram and thence to better selectivity and sensitivity.
Purification Process Design
The design of large-scale purification protocols should involve a minimal number of steps, since the recovery of a protein decreases as the number of purification steps increases. Standard purification methods involve pre-purification processes, including 1st separating the target protein from the cells expressing the protein. How this is accomplished depends on the expression system used to produce the target protein. Some expression systems express the protein intracellularly, necessitating the removal of the cells from the media, followed by lysing of the cells and subsequent purification of the protein. Other expression systems express the protein into the surrounding media, requiring the total media content to be passed through the initial purification column.
Concentration of the media feed stream containing the target protein is usually performed to lessen the amount of sample that is to be loaded onto the column. This requires time-consuming steps including filtration of the media to remove the cells expressing the protein. In addition, even after the concentration of the media, there is still a large volume that must be passed through the chromatographic support using a High Pressure Liquid Chromatography (HPLC) system (3). After the target protein is bound to the support, the column is washed with buffer to remove weakly bound proteins. Elution of the target protein is then performed, and the protein containing solution is neutralized, concentrated, and stored.
Thus, there is a need in the field of biological molecule separations including large scale chromatoghy for a ferromagnetic particle, as is in the field of cell selection, that exhibits andvantages over current technologies based on solid supports including superparamagnrtic particles. The advantages of FMP over other superparamagnetic particles include: cost, rapid reaction kinetics, rapid separation kinetics based on the ferromagnetic properties of the particles, simplicity of rinsing and adding of reagents, economy of reagents, and lower non-specific adherence. However, to date there has been no way of using ferromagnetic particles or density particles in biological molecule selection, nor have there been ferromagnetic or density particles useful in biological molecule selection. In this regard, while the term “dense particles” was used previously herein in reference to U.S. Pat. No. 5,576,185, for purposes of discussing the invention herein, the term “density particles” will be used. The reason for this, is that although such particles can have a greater density than the fluid in which used so that they separate by gravity, alternatively, such particles can have a relative density less than fluid so that separation occurs by floating or rising in the fluid. In both cases, density particles can be ferromagnetic or non-ferromagnetic as discussed hereafter.
There is also a need in the art for the elimination of the filtration and concentration steps thus enhancing the rate at which target protein(s) can be extracted and purified from a large-scale sample. The use of a particle that can be used within a sample for binding of the target protein, followed by rapid removal of the particle containing the bound protein, would result in rapid pre-process purification steps, which would greatly reduce the time required for purification. A particle that can be used in this manner would greatly reduce the time required for the pre process steps in the purification by eliminating the need to concentrate and filter the feed stream. For this reduction to occur, a particle would be needed that can be used as a chromatographic support in HPLC, be able to be modified so that affinity ligands can be covalently attached, and the particle must be able to mix with the target protein containing media and be separated from the cells and other debris in the protein containing feed stream. Removal of cells and other debris is essential for use in an HPLC system, since the debris can interfere in the operation of the system by building up pressure due to blocking of the flow by the debris. The following invention disclosed herein describes a unique method using unique FMP and or DP for accomplishing this task.